A major goal in any chemical or bioprocessing industry is the need to isolate and purify a product such as a protein from a complex mixture. Chromatography is well suited to a variety of uses in the field of biotechnology, since the technique is able to separate complex mixtures with great precision and is also suitable for more delicate products, such as proteins. Chromatographic separation matrices are designed to exploit the physical and chemical properties of the proteins of interest. For example, acidic proteins will interact and bind more strongly to ion exchange matrices having basic functionality and will therefore be retained by the matrix, and vice versa. The acidic proteins retained by the matrix can be subsequently removed or eluted by disrupting their interactions with the matrix. Another technique, affinity chromatography, exploits the unique biological specificity of a protein-ligand interaction. Affinity chromatography separations are not limited to protein purifications and the technique can be applied in principle where any particular ligand interacts specifically with a biomolecule. Substances that may be isolated by affinity chromatography include enzymes, antibodies, nucleic acids, hormones, etc. The concept is realised in practice by binding a ligand (or an ion exchange group) to an insoluble matrix. An impure mixture containing the protein to be separated is passed down a column packed with the matrix, where it will be adsorbed by the matrix. Adsorbed protein can then be eluted by altering the composition of the eluent so as to favour dissociation of the protein from the matrix.
Immobilisation of nucleophilic ligands, such as those containing amine, thiol or phenol groups, onto a solid support surface is conventionally performed by N-hydroxysuccinimide coupling or by direct opening of an epoxide immobilised onto the surface of the support (such as a gel) under aqueous conditions in presence of sodium hydroxide. Such methods can be successfully applied for the production of chromatographic media such as affinity matrices, where a low degree of substitution is required (typically ≦20 μmol/ml gel). In the case of ion exchange media, a higher degree of substitution is needed, suitably 80-150 μmol/ml. In this case, one of the methods of choice is based on the activation by bromination of immobilised allyl groups (see Scheme 1).
The use of bromine is hazardous and, in production, the use of a specific reactor that is dedicated to this type of reaction is often necessary. Furthermore, whilst the coupling procedure via bromination is efficient and mainly yields glycerol derivatives as side product, undesirable side products of the process may be observed including traces of unreacted allyl groups, together with a few percent of bromine atoms on the solid support.
Accordingly, there is still a need for improved methods for the synthesis of activated support materials suitable for the attachment of ligands for affinity and ion exchange chromatography. Sundberg, L. et al, (J. Chromatography, (1974), 90, 87-98) describe the use of bis-oxiranes for the introduction of reactive oxirane groups into agarose gels by reaction of the agarose with 1,4-butanediol diglycidyl ether in aqueous sodium hydroxide solution containing sodium borohydride. The method fulfilled the dual function of bridging the gel strands by cross-linking as well as providing functionality for subsequent attachment of ligands to the matrix. Matsumoto, I. et al, (J. Biochem., (1979), 85, 1091-98) describe inter alia the preparation of epoxy-activated SEPHAROSE™ 4B beads with epichlorhydrin, followed by derivatisation of the activated SEPHAROSE™ into amino- and carboxy-agarose. These methods are efficient for cases where a substitution level below 50 μmol/ml of gel is required, but are unsuitable for separation media with higher substitution levels.
Hjertén, S., et al, (J. Chromatography, (1986), 354, 203-210) describe a method for the preparation of agarose derivatives by first coupling γ-glycidoxypropyl-trimethoxysilane in an aqueous medium to agarose via the trimethoxy groups, followed by attachment of (for example) alcohols in acetone or dioxane solution in the presence of boron trifluoride diethyl etherate as a catalyst. However, the catalyst is corrosive and requires specific handling precautions. Furthermore, separation media prepared by this method following immobilisation of a ligand, are unstable under basic conditions (pH>10), due to the presence of the silane moiety. This is not compatible with the practical use of these media in the process purification of biomolecules where cleaning in situ procedures using high pH (>12) are routinely employed.
A difficulty that may arise when carrying out nucleophilic substitution in heterogeneous reaction systems is that the reactants may not mix effectively. In such nucleophilic substitution reactions, the substrate is usually insoluble in water or other polar solvent, while the nucleophile is often an anion soluble in water but not in the organic solvent or substrate. Consequently, when the two reactants are brought together, their concentrations in the same phase are too low for convenient reaction rates. One way to overcome this is to use a solvent that will dissolve both species. Another way is to use a two phase system with a phase transfer catalyst. The present invention provides an alternative method for immobilising ligands and activating solid support materials with epoxy groups.